Developmental genetics of mutants that specify knotted leaves in maize

Of seven dominant knotted-leaf mutants tested, six mapped at or near Kn1 on the long arm of chromosome 1, and one was not linked to Kn1. Comparisons of phenotypes among these mutants allowed us to focus on a systematic abnormality: the parenchyma cells associated with lateral veins do not fully differentiate into bundle sheath, mesophyll or upper sclerenchyma. The more dramatic expression of Kn1 mutants—knots, ligule alterations and twisting—is sporadic and dependent on the time when the mutant acts in leaf primordium development. Using lw to mark leaf sectors that lose Kn1 following X-irradiation, we show that the knotted-leaf phenotype encoded by chromosome 1L is autonomous. Analysis of sectors lacking a particular Kn1 gene ( Kn1-N2) suggests that Kn1 itself, rather than a linked modifier gene, is autonomous in the leaf primordium. Aneuploid studies using various translocations involving 1L and marked by Adh1 allozymes are compared. The Kn1 mutant appears to encode a "new" function or a considerable overproduction of an extant product in the leaf. Kn1/- 1L hypoploids either express knotted poorly or not at all; transvection is ruled out, but the cause for this modification of Kn1 expression is not yet known.—Our working hypothesis is that Kn1 mutants permit the expression of a product that is usually not produced in leaf primordial cells. We suggest that this product interferes with the early cell-type commitments of cells near lateral veins. Thus, relatively uncommitted cells are present in more mature blades, where they may divide unexpectedly into knots or may induce bits of ligule.     

Etioplast-chloroplast transformation in maize leaves: Effects of tissue age and light intensity

Summary The effects of light intensity and cell age on the greening of etioplasts were studied in seedlings of maize.We could see that in the youngest tissues examined by us the etioplast greening is very fast and occurs according to a particular pattern which is characterized by the contemporary presence of grana and large non crystalline prolamellar bodies. On the contrary, in the oldest examined tissues the etioplast greening is slow and the formation of grana appears to be delayed and subsequent to the using up of the prolamellar bodies.In the young tissues the intensity of the light mainly affects the duration of the lag-phase preceding the chlorophyll accumulation, while in the old tissues it also affects the total amount of chlorophyllous pigments, the restraining effect of the light appearing amplified by a concomitant restraining effect of cell age.Supported by a grant of C.N.R.     

Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension

The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.     

Glycolate oxidase isoforms are distributed between the bundle sheath and mesophyll tissues of maize leaves

Glycolate oxidase (EC 1.1.3.15) activity was detected both in the bundle sheath (79%) and mesophyll (21%) tissues of maize leaves. Three peaks of glycolate oxidase activity were separated from maize leaves by the linear KCl gradient elution from the DEAE-Toyopearl column. The first peak corresponded to the glycolate oxidase isoenzyme located in the bundle sheath cells, the second peak had a dual location and the third peak was related to the mesophyll fraction. The mesophyll isoenzyme showed higher affinity for glycolate (Km 23 micromol x L(-1)) and a higher pH optimum (7.5-7.6) as compared to the bundle sheath isoenzyme (Km 65 micromol x L(-1), pH optimum 7.3). The bundle sheath isoenzyme was strongly activated by isocitrate and by succinate while the mesophyll isoenzyme was activated by isocitrate only slightly and was inhibited by succinate. It is concluded that although the glycolate oxidase activity is mainly attributed to the bundle sheath, conversion of glycolate to glyoxylate occurs also in the mesophyll tissue of C4 plant leaves.     

Ectopic expression of maize knotted1 results in the cytokinin-autotrophic growth of cultured tobacco tissues

The ectopic expression of knotted homologues has cytokinin-like effects on plant morphology. The functional relationship between knotted and cytokinins was investigated in cultures of leaf tissue established from tobacco (Nicotiana tabacum L. cv. Havana 425) plants transformed with the maize knotted1 (kn1) gene regulated by cauliflower mosaic virus 35S RNA expression signals. In contrast to leaf tissues of untransformed plants, leaf tissues of kn1 transformants were capable of sustained, cytokinin-autotrophic growth on auxin-containing medium and resembled the tobacco cytokinin-autotrophic mutants Hl-1 and Hl-2. The concentration of 18 cytokinins was measured in cultures initiated from leaves of three independent kn1 transformants and the Hl-1 and Hl-2 mutants. Although cytokinin contents were variable, the content of several cytokinins in Kn1, Hl-1 and Hl-2 tissue lines was at least 10-fold higher than that of wild-type tobacco tissues and in the range reported for other cytokinin-autotrophic tobacco tissues. These results suggest that the cytokinin-autotrophic growth of Kn1 lines could result from elevated steady-state levels of cytokinins. Received: 7 July 1999 / Accepted: 10 November 1999     

Bundle sheath cells of small veins in maize leaves are the location of uptake from the xylem.

Rb(+) as a tracer for K(+) was used to test the hypothesis that uptake of K(+) from xylem vessels of small veins into the symplast of maize leaves occurs at the xylem/bundle sheath cell interface. 22.5 min after immersing cut leaves into 20 mM RbCl+1 mM KCl, Rb(+) appeared in the cells of the leaves. Sections of these leaves were freeze-dried. In cryo-thin sections (5 microm), (85)Rb(+) and (41)K(+) content was determined by laser microprobe mass analysis with a large resolution of about 1 microm. Determining the ratio of (85)Rb(+) to (41)K(+) in the cell walls and cytosols of bundle sheath cells, mesophyll cells, and in the cells between the xylem elements resulted in the following picture: In small veins, Rb(+) entered the symplast directly at the xylem/bundle sheath cell interface.     

Appropriate characters for racial classification in maize

To determine the relative importance of the genotype, the environment, and their interaction on the expression of morphological characters in maize races, 50 Mexican races and 24 races from Central America, South America, and the U.S. were grown in several locations and seasons in México and 47 characters were measured directly. Estimates of the ratio of variance components, rc = [Vc/(Ve+VCs/], were used as criteria to determine the appropriate characters for racial classification. Twenty-four useful variables were identified. Analysis of the structure of the data matrix facilitated visual examination of correlations among the variables and of the variability represented by each variable. Based on these analyses, a minimum list of 9 characters was suggested to be appropriate variables for racial classification: number of leaves per plant, branched part length/tassel length, central spike internode length, male glume length, kernel width, rachis segment length, pith diameter, ear diameter/length, and kernel width/length.     

Acetyl-coenzyme A carboxylase in maize leaves

Purified chloroplasts from mesophyll and bundle sheath cells of maize leaves have been shown to be the location of acetyl-CoA carboxylase. In disrupted chloroplasts the enzyme was recovered in the stromal fraction, along with protein-bound biotin; acetyl-CoA carboxylase activity did not require a membrane component. Mg²⁺ and ATP are required for activity and sulfhydryl protecting agents enhance stability of the enzyme. Acetyl-CoA carboxylase activity was independent of leaf development in cell-free extracts of maize. Comparison of acetyl-CoA carboxylase activity with [¹⁴C]acetate incorporation into lipids, in isolated chloroplasts from developing leaves of maize, indicate that acetyl-CoA carboxylase is not limiting fatty acid synthesis.     

Succinyl-CoA synthetase in greening maize leaves

In extracts of greening maize leaves succinyl-CoA synthetase was present in both a particulate and a soluble fraction. Aqueous and non-aqueous fractionation together with determination of chlorophyll content and cytochrome oxidase activity indicated that the enzyme was neither located, nor originated in plastids. Pre-illumination of leaves caused only small increases in the activity of either the particulate or the soluble enzyme. The soluble enzyme was ATP specific and had a low affinity for succinate (Km = 63 mM).     

Development of aleurone and sub-aleurone layers in maize

Electron-microscope studies indicate that the aleurone tissue of maize (Zea mays L.) starts developing approximately 10–15 days after pollination in stocks that take ca. 40 days for the aleurone to mature completely. Development commences when specialized endosperm cells adjacent to the maternal nucellar layer start to differentiate. Differentiation is characterized by the formation of aleurone protein bodies and spherosomes. The protein bodies of the aleurone layer have a vacuolar origin whereas the protein bodies of the immediate underlying endosperm cells appear to develop from protrusions of the rough endoplasmic reticulum. Thus, two morphologically and developmentally distinct types of protein bodies are present in these adjacent tissues. The spherosomes of the aleurone layer form early in the development of this tissue and increase in number as the tissue matures. During the final stages of maturation, these spherosomes become closely apposed to the aleurone grains and the plasma membrane. No further changes are apparent in the structure of the aleurone cells after 40 days from pollination when the caryopsis begins to desiccate.     

Regulation of phosphoenolpyruvate carboxylase activity in maize leaves

The aim of this work was to investigate how light regulates the activity of phosphoenolpyruvate carboxylase in vivo in C₄ plants. The properties of phosphoenolpyruvate carboxylase were investigated in extracts which were rapidly prepared (in less than 30 seconds) from darkened and illuminated leaves of Zea mays. Illumination resulted in a significant decrease in the S_0.5(phosphoenolpyruvate) but there was no change in V_max. The form of the enzyme from illuminated leaves was less sensitive to malate inhibition than was the form from darkened leaves. At low concentrations of phosphoenolpyruvate, the activity of the enzyme was strongly stimulated by glucose-6-phosphate, fructose-6-phosphate, triose-phosphate, alanine, serine, and glycine and was inhibited by organic acids. The enzyme was assayed in mixtures of metabolites at concentrations believed to be present in the mesophyll cytosol in the light and in the dark. It displayed low activity in a simulated `dark' cytosol and high activity in a simulated `light' cytosol, but activities were different for the enzyme from darkened compared to illuminated leaves.     

稻纵卷叶螟幼虫在卷叶内的栖居特性

稻纵卷叶螟Cnaphalocrocismedinalis,幼虫在卷叶内栖居特性田间调查研究表明,盛孵期后3d,1～2龄幼虫新卷叶有虫率为100%,旧卷叶有虫率为0～2857%,平均为1225%;盛孵期后5d,1～3龄幼虫新卷叶有虫率为50%～9286%,平均为6902%,旧卷叶有虫率为0～32%,平均为1128%。因此,稻纵卷叶螟1～3龄幼虫在田间主要栖居于新、旧卷叶内,田间药效评价时应以新、旧卷叶内幼虫量为依据计算防治效果。     

Lipid biosynthesis in green leaves of developing maize

Successive leaf sections from the base to the tip of rapidly developing leaves of 7-day-old maize (Zea mays var. Kelvedon Glory), grown in the light, utilized acetate for fatty acid biosynthesis in a very divergent manner. Basal regions of the leaf containing proplastids synthesized insignificant proportions of unsaturated fatty acids and appreciable proportions of fatty acids with 20 or more carbon atoms. An increase in the light intensity during incubations with acetate-1-¹⁴C resulted in very little enhancement of either total or polyunsaturated fatty acid biosynthesis in this tissue.     

Prior illumination and the respiration of maize leaves in the dark

The course of respiration of attached maize (Zea mays L.) leaves was measured by infrared gas analysis of CO₂ efflux in the dark following illumination in atmospheres of 300 microliters of CO₂ per liter of air, CO₂-free air, and CO₂-free N₂ containing 400 microliters of O₂ per liter. CO₂ efflux from control leaves started 3 to 4 minutes after darkening, increased to a maximum after about 20 minutes, and returned to a steady minimum after 2 to 3 hours. Respiration was quantitatively related to prior illumination, independent of net CO₂ fixation in the light, and depressed by N₂. Light, but not air, was required to produce a substrate for respiration in the subsequent dark period; air was required for oxidation of the substrate to CO₂. The stimulation of respiration by prior illumination in maize leaves differs in its slower onset and greater duration from the postillumination burst of photorespiration.     

Growth-induced water potentials and the growth of maize leaves

Profiles of water potential (Psi(w)) were measured from the soil through the plant to the tip of growing leaves of fully established maize (Zea mays L.). The profiles revealed gradients in transpiration-induced Psi(w) extending upward along the transpiration path, and growth-induced Psi(w) extending radially between the veins in the elongating region of the leaf base. Water moving upward required a small gradient while that moving radially required a much larger gradient primarily because the protoxylem vessels were encased in many small, undifferentiated cells that were likely to act as a barrier to radial flow. Upon maturation, these small cells enlarged and some began to conduct water, probably decreasing the barrier. In the mature leaf, the growth-induced Psi(w) were absent but the transpiration-induced Psi(w) remained. When leaves were growing, the growth-induced Psi(w) moved water into the elongating cells during the day and night, and it shifted with changes in transpiration-induced Psi(w). The shift involved solutes accumulating in the growing region. When water was withheld, the growth-induced Psi(w) disappeared and leaf elongation ceased even though turgor pressure was at its highest. Turgor was maintained by osmotic adjustment that doubled the osmotic potential of the elongating cells. If elongation resumed at night or with rewatering, the growth-induced Psi(w) reappeared. If pressure was applied to the soil/root system to cause guttation and re-establish the growth-induced Psi(w), elongation resumed immediately. These findings support the hypothesis that the primary control of growth is the disappearance and reappearance of the growth-induced Psi(w) because the potential changed in the xylem and nearby cells, blocking or permitting radial water movement and thus blocking or permitting growth.     

Nitrogen metabolism in the stalk tissue of maize

During ear development in maize (Zea mays L.), nitrogenous compounds are translocated from vegetative organs to the kernels. At anthesis, the stalk contains approximately 40% of the total plant N, and contributes 45% of the N remobilized to the ear. Therefore, the stalk has an important function as a temporary reservoir for N. Little is known of the metabolism of maize stalks, and this paper describes initial studies of enzymes of N metabolism. High in vitro activity of glutamine synthetase (GS) in maize stalk samples throughout ear development contrasted with a peak in activity of glutamate synthase soon after anthesis and negligible nitrate reductase. With fresh sections of stalk tissue collected at anthesis, ¹⁵N-feeding experiments confirmed high GS and low nitrate reductase activities. Two isoforms of GS were separated from extracts from stalk tissue: GS1, the cytoplasmic form, increased to maximum levels at 2 weeks postanthesis and remained fairly high thereafter; whereas the plastidic form, GS2, declined progressively during kernel development. Western blot analysis confirmed the presence of constantly high levels of GS protein after anthesis. The levels of GS proteins decreased after transfer of N-starved, hydroponically grown plants to N-rich conditions in order to restrict remobilization of N. In contrast, transfer of plants grown under abundant N conditions to N-free medium, which encourages N remobilization, resulted in a relative increase in GS protein. Because glutamine is the major form of N transported in maize, the results indicate that GS, specifically the GS1 isoform, has a central role in the remobilization on nitrogenous compounds from the stalk to the ear.     

tie-dyed1 Regulates carbohydrate accumulation in maize leaves

Acquisition of cell identity requires communication among neighboring cells. To dissect the genetic pathways regulating cell signaling in later leaf development, a screen was performed to identify mutants with chloroplast pigmentation sectors that violate cell lineage boundaries in maize (Zea mays) leaves. We have characterized a recessive mutant, tie-dyed1 (tdy1), which develops stable, nonclonal variegated yellow and green leaf sectors. Sector formation requires high light, occurs during a limited developmental time, and is restricted to leaf blade tissue. Yellow tdy1 sectors accumulate excessive soluble sugars and starch, whereas green sectors appear unaffected. Significantly, starch accumulation precedes chlorosis in cells that will become a yellow sector. Retention of carbohydrates in tdy1 leaves is associated with a delay in reproductive maturity, decreased stature, and reduced yield. To explain the tdy1 sectoring pattern, we propose a threshold model that incorporates the light requirement and the hyperaccumulation of photoassimilates. A possible function consistent with this model is that TDY1 acts as a sugar sensor to regulate an inducible sugar export pathway as leaves develop under high light conditions.     

Reduction of paraquat toxicity in maize leaves by benzyladenine

The protective effect of a cytokinin benzyladenine (BA), against toxicity of paraquat (PQ), a widely used herbicide and a well-known oxidative stress inducer, was investigated in the leaves of maize. Maize leaves have been pretreated with BA at concentrations of 1, 10 and 100 microM and afterwards treated with PQ. At all concentrations tested, BA retarded PQ-induced decreases in chlorophyll, carotenoid and ascorbic acid contents. Pretreatment with 10 and 100 microM of BA significantly increased superoxide dismutase (SOD) activity after 8 h of PQ treatment but there was no significant change in SOD activity in the leaves pretreated with BA at 12 and 24 h. However, peroxidase activity significantly increased in 100 microM of BA pretreated leaves. Results indicate that pretreatment with BA reduce PQ toxicity and BA-treated plants might become more tolerant against oxidative stress.